PROJECTSUMMARY Tauisamicrotubule-stabilizingproteinthatisabundantinneurons.Itisahighlysoluble,intrinsicallydisordered protein(IDP)withlittletendencyforaggregationundernativeconditions.However,underseveralexperimental conditionsandinavarietyofneurodegenerativedisordersincludingAlzheimer?sdisease,Taucanspreadfrom cell to cell and aggregates as intra-cellular ?-sheet fibrilar deposits. Our laboratories have critical new data concerningthetemporal,structuralandcellbiologicaldetailsofTaumisfoldingandfluid-phaseassembly?the basis of this proposal. Our research team consists of a cell biologist, a physical chemist, and a theoretical biophysicist.Workingtogethercloselyinaniterativemannerweintendtodeterminethepathwayfromnormal Tautodisease-relatedTaufibrils.Thetoolsforthisanalysisinclude(a)cellularsystemscapableofaddressing invivoTauinteractions,andindirectlyitsconformationalstatebasedonavarietyofmolecularprobes;?(b)site- directedspinlabeling,electronparamagneticresonance(EPR)lineshapeanalysisandpulseddipolarEPRto determine conformational signatures of Tau;? and (c) fully atomistic modeling of IDP conformations, their populationsandenergetics,andcoarse-grainedsimulationofhigher-orderassembliesofTau.Theconceptual flowoftheproposalbeginswitharemarkableobservationfromtheHanlab:Whenexposedtosub-stoichiometric amountsofheparin,segmentsofTaudramaticallyextendbyananometertosolvent-exposethehydrophobic PHF6(*) segment capable of stacking into neat ?-sheets. This observation correlates with the appearance of fibrils,andthuswerefertothisinitiatingstepas?onpathway?seeding.Invivo,Tauisknowntopopulateavast conformationallandscapecontrolledbyalternativesplicing,mutationsandpost-translationalmodifications.We propose that the IDP Tau populates an ensemble of different conformations with different aggregation propensities, fibril morphologies and interaction partners, depending on the exact Tau variant. However, the defining and specific conformational signatures within this ensemble are unknown. Determining the conformationalsignaturesofaggregation-proneTauvariantsisourcoreobjective,whileamissingpuzzlepiece inconnectingTauconformationtocellularinteractionsistheexistenceandnatureofaggregationintermediates. Inthisvein,theHanlabdiscoveredthatRNAinducesliquid-liquidphaseseparationofTauinvitrointoprotein dropletsheldtogetherbyweakelectrostaticforces.Attheinvivocellularlevel,theKosiklabdiscoveredTau- tRNAcomplexes,therebyaddingTautothegrowinglistofRNA-bindingproteinsinvolvedinneurodegeneration, andcapableofestablishingliquid-liquidphaseseparationinthecytoplasm.TheTau-tRNAcomplexesmaybea physiologic or pathological entity?we will obtain clues by determining their loci in neuronal cells. Finally, we intendtolearnwhethertheconformationofTau,asmodulatedbydiseasemutationsorco-factors,influences thestabilityandinvivolocalityoftheTau-tRNAcomplexes.Ourgoalistodiscoveradetailedroutefromsoluble Tautofibrils,fromthenanometertothecellularlevel,anddiscoverthepathologicalentitiesofTauaggregation.